人HCN4基因真核表达质粒构建及在HEK293细胞中的表达

Construction of human HCN4 gene eukaryon expression vector and its expression in HEK293 cells

目的:构建人HCN4基因的真核表达质粒并转染人胚胎肾细胞,建立异源性表达HCN4通道模型。方法:通过聚合酶链反应扩增人HCN4基因全长核苷酸序列,双酶切(Xho I和EcoRI)后插入真核表达质粒p IRES2-EGFP中,构建重组真核表达质粒p IRES2-HCN4-EGFP。应用脂质体法将重组质粒p IRES2-HCN4-EGFP转染入HEK293细胞中进行表达。全细胞膜片钳技术检测HCN4通道电流。结果:酶切及测序的结果显示p IRES2-HCN4-EGFP构建成功,倒置荧光显微镜能观察到EGFP绿色荧光,反转录-聚合酶链反应检测到HCN4 mRNA表达,膜片钳检测到HCN4基因编码的通道电流。结论:成功建立异源性表达HCN4通道模型,为进一步研究HCN4通道电生理特性提供了实验基础。

Objective：To construct eukaryon expression vector of human HCN4 gene and transfect it into human embryonic kidney cells （HEK293）. Methods：cDNA encoding human HCN4 gene was amplified by polymerase chain reaction,then digested with the restriction endonucleases Xho1 and EcoR1 and inserted into eukaryotic expressing vector pIRES2-EGFP that was transfected into HEK293 cells by Fugene HD.Whole-cell patch clamp indicated that hHCN4 gene was transfected into HEK293cells.Results：Double enzyme digestion and DNA sequencing confirmed that the HCN 4 gene was completely ac-curate.GFP was observed in the transfected 293 cells under a fluorescent microscope.HCN4 mRNA expression was confirmed by RT-PCR.Whole-cell patch clamp recorded ionic currents of transfected HCN4.Conclusion：The pIRES-HCN4-EGFP eukaryon expression vector was successfully constructed,and pacemaker channel HCN4 gene heterologous expression was established.